Combination therapy with an anti-her2 antibody-drug conjugate and a bcl-2 inhibitor

ABSTRACT

The present invention is directed to a combination therapy involving an anti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor for the treatment of a patient suffering from cancer, particularly, a HER2-expressing cancer.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 28, 2016, isnamed GNE-0416-WO.txt and is 6,298 bytes in size.

TECHNICAL FIELD

The present invention is directed to a combination therapy involving ananti-HER2 antibody-drug conjugate and a Bcl-2 inhibitor for thetreatment of cancer. In a particular embodiment, the invention concernsmethods of using trastuzumab-MCC-DM1 (trastuzumab emtansine; KADCYLA®)and a selective Bcl-2 inhibitor for the treatment of HER2-positivecancer, such as HER2-positive breast cancer or gastric cancer.

BACKGROUND

Anti-HER2 Antibody-Drug Conjugates

The HER2 (ErbB2) receptor tyrosine kinase is a member of the epidermalgrowth factor receptor (EGFR) family of transmembrane receptors.Overexpression of HER2 is observed in approximately 20% of human breastcancers (hereinafter referred to as HER2-positive breast cancer) and isimplicated in the aggressive growth and poor clinical outcomesassociated with these tumors (Slamon et al (1987) Science 235:177-182).HER2 protein overexpression can be determined using animmunohistochemistry based assessment of fixed tumor blocks (Press M F,et al (1993) Cancer Res 53:4960-70).

Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2,Genentech) is a recombinant DNA-derived, IgG1 kappa, monoclonal antibodythat is a humanized version of a murine anti-HER2 antibody (4D5) thatselectively binds with high affinity in a cell-based assay (Kd=5 nM) tothe extracellular domain of HER2 (U.S. Pat. Nos. 5,677,171; 5,821,337;6,054,297; 6,165,464; 6,339,142; 6,407,213; 6,639,055; 6,719,971;6,800,738; 7,074,404; Coussens et al (1985) Science 230:1132-9; Slamonet al (1989) Science 244:707-12; Slamon et al (2001) New Engl. J. Med.344:783-792). Trastuzumab has been shown, in both in vitro assays and inanimals, to inhibit the proliferation of human tumor cells thatoverexpress HER2 (Hudziak t al (1989) Mol Cell Biol 9:1165-72; Lewis etal (1993) Cancer Immunol Immunother; 37:255-63; Baselga et al (1998)Cancer Res. 58:2825-2831). Trastuzumab is a mediator ofantibody-dependent cellular cytotoxicity, ADCC (Lewis et al (1993)Cancer Immunol Immunother 37(4):255-263; Hotaling et al (1996)[abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471; Pegram MD, et al (1997) [abstract]. Proc Am Assoc Cancer Res; 38:602; Sliwkowskiet al (1999) Seminars in Oncology 26(4), Suppl 12:60-70; Yarden Y. andSliwkowski, M. (2001) Nature Reviews: Molecular Cell Biology, MacmillanMagazines, Ltd., Vol. 2:127-137).

HERCEPTIN® was approved in 1998 for the treatment of patients withHER2-overexpressing metastatic breast cancers (Baselga et al, (1996) J.Clin. Oncol. 14:737-744) that have received extensive prior anti-cancertherapy, and has since been used in over 300,000 patients (Slamon D J,et al. N Engl J Med 2001; 344:783-92; Vogel C L, et al. J Clin Oncol2002; 20:719-26; Marty M, et al. J Clin Oncol 2005; 23:4265-74; Romond EH, et al. T N Engl J Med 2005; 353:1673-84; Piccart-Gebhart M J, et al.N Engl J Med 2005; 353:1659-72; Slamon D, et al. [abstract]. BreastCancer Res Treat 2006, 100 (Suppl 1): 52). In 2006, the FDA approvedHERCEPTIN® (trastuzumab, Genentech Inc.) as part of a treatment regimencontaining doxorubicin, cyclophosphamide and paclitaxel for the adjuvanttreatment of patients with HER2-positive, node-positive breast cancer.

An alternative approach to antibody-targeted therapy is to utilizeantibodies for delivery of cytotoxic drugs specifically toantigen-expressing cancer cells. Antibody-drug conjugates, or ADCs, aremonoclonal antibodies to which highly potent cytotoxic agents have beenconjugated. ADCs represent a novel approach to conferring tumorselectivity on systemically administered anti-tumor therapeutics.Utilizing surface antigens that are tumor-specific and/or overexpressed,ADCs are designed to focus the delivery of highly potent cytotoxicagents to tumor cells. The potential of this approach is to create amore favorable therapeutic window for such agents than could be achievedby their administration as free drugs.

Maytansinoids, derivatives of the anti-mitotic drug maytansine, bind tomicrotubules in a manner similar to vinca alkaloid drugs (Issell B F etal (1978) Cancer Treat. Rev. 5:199-207; Cabanillas F et al. (1979)Cancer Treat Rep, 63:507-9. DM1 is a thiol-containing maytansinoidderived from the naturally occurring ester ansamitocin P3 (Remillard S,Rebhun L I, Howie G A, et al. (1975) Science 189(4207):1002-1005.3;Cassady J M, Chan K K, Floss H G. (2004) Chem Pharm Bull 52(1):1-26.4).The related plant ester, maytansine, has been studied as achemotherapeutic agent in approximately 800 patients, administered at adose of 2.0 mg/m2 every 3 weeks either as a single dose or for 3consecutive days (Issell B F, Crooke S T. (1978) Maytansine. CancerTreat Rev 5:199-207). Despite preclinical activity, the activity ofmaytansine in the clinic was modest at doses that could be safelydelivered. The dose-limiting toxicity (DLT) was gastrointestinal,consisting of nausea, vomiting, and diarrhea (often followed byconstipation). These toxicities were dose dependent but not scheduledependent. Peripheral neuropathy (predominantly sensory) was reportedand was most apparent in patients with preexisting neuropathy.Subclinical transient elevations of hepatic transaminase, alkalinephosphatase, and total bilirubin were reported. Constitutionaltoxicities, including weakness, lethargy, dysphoria, and insomnia, werecommon. Less common toxicities included infusion-site phlebitis and mildmyelosuppression. Further development of the drug was abandoned in the1980s because of the narrow therapeutic window.

Trastuzumab-MCC-DM1 (T-DM1, trastuzumab emtansine, ado-trastuzumabemtansine, KADCYLA®), a novel antibody-drug conjugate (ADC) for thetreatment of HER2-positive breast cancer, is composed of the cytotoxicagent DM1 (a thiol-containing maytansinoid anti-microtubule agent)conjugated to trastuzumab at lysine side chains via an MCC linker, withan average drug load (drug to antibody ratio) of 3.5. After binding toHER2 expressed on tumor cells, T-DM1 undergoes receptor-mediatedinternalization, resulting in intracellular release of cytotoxicDM1-containing catabolites and subsequent cell death.

In a Phase I study of T-DM1 (TDM3569g), the maximum tolerated dose (MTD)of T-DM1 administered by IV infusion every 3 weeks (q3w) was 3.6 mg/kg.A DLT (Dose-Limiting Toxicity) consisted of transient thrombocytopeniain patients treated at 4.8 mg/kg. Treatment with 3.6 mg/kg q3w was welltolerated and associated with significant clinical activity. (Krop(2010) J. Clin. Oncol. 28(16):2698-2704). That same study also showedthat weekly dosing with 2.4 mg/kg was also well tolerated and hadanti-tumor activity. (Beeram (2012) Cancer 118(23):5733-5740.)

A Phase II study (TDM4374g) demonstrated that T-DM1, administered at 3.6mg/kg q3w, had single-agent anti-tumor activity in a heavily pre-treatedpatient population having HER2-positive metastatic breast cancer. (Krop(2012) 30(26):3234-3241.) A Phase III study (TDM4370g) demonstrated thatT-DM1, administered at 3.6 mg/kg q3w, significantly prolongedprogression-free survival and overall survival with less toxicitycompared to treatment with lapatinib plus capecitabine in patients withHER2-positive advanced breast cancer previously treated with trastuzumaband a taxane. (Verma (2012) New England Journal of Medicine367:1783-1791.)

The U.S. Food and Drug Administration approved ado-trastuzumabemtansine, marketed under the tradename KADCYLA®, on Feb. 22, 2013 forthe treatment of patients with HER2-positive, metastatic breast cancerwho previously received treatment with trastuzumab and a taxane.

Bcl-2 Inhibitors

The Bcl-2 family of proteins regulates programmed cell death triggeredby developmental cues and in response to multiple stress signals (Cory.S., and Adams, J. M., Nature Reviews Cancer 2 (2002) 647-656; Adams,Genes und Development 17 (2003) 2481-2495; Danial, N. N., and Korsmeyer,S. J., Cell 116 (2004) 205-219). Whereas cell survival is promoted byBcl-2 itself and several close relatives (Bcl-xL, Bcl-W, Mcl-1 and A1),which bear three or four conserved Bcl-2 homology (BH) regions,apoptosis is driven by two other sub-families. The initial signal forcell death is conveyed by the diverse group of BH3-only proteins,including Bad, Bid, Bim, Puma and Noxa, which have in common only thesmall BH3 interaction domain (Huang and Strasser, Cell 103 (2000)839-842). However, Bax or Bak, multi-domain proteins containing BH1-BH3,are required for commitment to cell death (Cheng, et al., Molecular Cell8 (2001) 705-711; Wei, M. C., et al., Science 292 (2001) 727-730; Zong,W. X., et al., Genes and Development 15 148 (2001) 1-1486). Whenactivated, they can permeabilize the outer membrane of mitochondria andrelease pro-apoptogenic factors (e.g. cytochrome C) needed to activatethe caspases that dismantle the cell (Wang, K., Genes and Development 15(2001) 2922-2933; (Adams, 2003 supra); Green, D. R., and Kroemer, G.,Science 305 (2004) 626-629).

Interactions between members of these three factions of the Bcl-2 familydictate whether a cell lives or dies. When BH3-only proteins have beenactivated, for example, in response to DNA damage, they can bind viatheir BH3 domain to a groove on their pro-survival relatives (Sattler,et al., Science 275 (1997) 983-986). How the BH3-only and Bcl-2-likeproteins control the activation of Bax and Bak, however, remains poorlyunderstood (Adams, 2003 supra). Most attention has focused on Bax. Thissoluble monomeric protein (Hsu, Y. T., et al., Journal of BiologicalChemistry 272 (1997) 13289-13834; Wolter, K. G., et al., Journal of CellBiology 139 (1997) 1281-92) normally has its membrane targeting domaininserted into its groove, probably accounting for its cytosoliclocalization (Nechushtan, A., et al., EMBO Journal 18 (1999) 2330-2341;Suzuki, et al., Cell 103 (2000) 645-654; Schinzel, A., et al., J CellBiol 164 (2004) 1021-1032). Several unrelated peptides/proteins havebeen proposed to modulate Bax activity, reviewed in Lucken-Ardjomande,S., and Martinou, J. C., J Cell Sci 118 (2005) 473-483, but theirphysiological relevance remains to be established. Alternatively, Baxmay be activated via direct engagement by certain BH3-only proteins(Lucken-Ardjomande, S., and Martinou, J. C, 2005 supra), the bestdocumented being a truncated form of Bid, tBid (Wei, M. C., et al.,Genes und Development 14 (2000) 2060-2071; Kuwana, T., et al., Cell 111(2002) 331-342; Roucou, X., et al., Biochemical Journal 368 (2002)915-921; Cartron, P. F., et al., Mol Cell 16 (2004) 807-818). Asdiscussed elsewhere (Adams 2003 supra), the oldest model, in which Bcl-2directly engages Bax (Oltvai, Z. N., et al., Cell 74 (1993) 609-619),has become problematic because Bcl-2 is membrane bound while Bax iscytosolic, and their interaction seems highly dependent on thedetergents used for cell lysis (Hsu, Y. T., and Youle, 1997 supra).Nevertheless, it is well established that the BH3 region of Bax canmediate association with Bcl-2 (Zha, H., and Reed, J., Journal ofBiological Chemistry 272 (1997) 31482-88; Wang, K., et al., Molecularund Cellular Biology 18 (1998) 6083-6089) and that Bcl-2 prevents theoligomerization of Bax, even though no hctcrodimcrs can be detected(Mikhailov, V., et al., Journal of Biological Chemistry 276 (2001)18361-18374). Thus, whether the pro-survival proteins restrain Baxactivation directly or indirectly remains uncertain.

Although Bax and Bak seem in most circumstances to be functionallyequivalent (Lindsten, T., et al., Molecular Cell 6 (2000) 1389-1399;Wei, M. C., et al., 2001 supra), substantial differences in theirregulation would be expected from their distinct localization in healthycells. Unlike Bax, which is largely cytosolic, Bak resides in complexeson the outer membrane of mitochondria and on the endoplasmic reticulumof healthy cells (Wei, M. C., et al., 2000 supra; Zong, W. X., et al.,Journal of Cell Biology 162 (2003) 59-69). Nevertheless, on receipt ofcytotoxic signals, both Bax and Bak change conformation, and Baxtranslocates to the organellar membranes, where both Bax and Bak thenform homo-oligomers that can associate, leading to membranepermeabilization (Hsu, Y. T., et al., PNAS 94 (1997) 3668-3672; Wolter,K. G., et al., 1997 supra; Antonsson, B., et al., Journal of BiologicalChemistry 276 (2001) 11615-11623; Nechushtan, A., et al., Journal ofCell Biology 153 (2001) 1265-1276; Wei, M. C., et al., 2001 supra;Mikhailov, V., et al., Journal of Biological Chemistry 278 (2003)5367-5376).

There exist various Bcl-2 inhibitors, which all have the same propertyof inhibiting prosurvival members of the Bcl-2 family of proteins andare therefore promising candidates for the treatment of cancer. SuchBcl-2 inhibitors are e.g. Oblimersen, SPC-2996, RTA-402, Gossypol,AT-101, Obatoclax mesylate, A-371191, A-385358, A-438744, ABT-737,ABT-263 (navitoclax), AT-101, BL-11, BL-193, GX-15-003,2-Methoxyantimycin A₃, HA-14-1, KF-67544, Purpurogallin, TP-TW-37,YC-137 and Z-24, and are described e.g. in Zhai, D., et al., Cell Deathand Differentiation 13 (2006) 1419-1421.

The link between other the Bcl-2 family proteins and cancer is also wellestablished and amply documented (Strasser, A. 2011 EMBO J. 30,3667-3683), and inhibitors of other Bcl family members are also known.Bcl-X_(L)-selective inhibitors A-1155463 and A-1331852 are described,for example, in Leverson et al., Science Translational Medicine Vol 7,Issue 279 279ra40. Selective benzothiazole hydrazone inhibitors ofBcl-X_(L) are disclosed in Sleebs et al., J. Med. Chem. 2013, 56,5514-5540. For the description of other Bcl-X_(L) inhibitors see, e.g.Koehler et al., ACS Med. Chem. Lett. 2014, 5, 662-667; and Tao et al,ACS Med. Chem. Lett. 2014, 5, 1088-10. MCl-1 inhibitors and their usesas cancer therapeutics are described, for example, in Leverson et al.,Cell Death and Disease (2015) 6, e1590; Bruncko et al., J. Med. Chem.2015, 58, 2180-2194; Petros et al., Boorganic & Medicinal ChemistryLetters 24 (2014) 1484-1488; Abulwerdi et al., Mol Cancer Ther 2014;13:565-5; Abuilwerdi et al., J. Med. Chem. 2014, 57, 4111-4133; Burke etal., J. Med. Chem. 2015, 58, 3794-3805; Friberg et al., J. Med. Chem.2013, 56, 15-30; and belmar et al., Pharmacology & Theraputics 145(2015) 76-84. Mcl-1/Bcl-xL dual inhibitors are disclosed by Tanaka etal., J. Med. Chem. 2013, 56, 9635-9645.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method for the treatment of acancer in a human in need thereof comprising administering to such humanan effective amount of an anti-HER2 antibody-drug conjugate and aninhibitor of a Bcl family protein.

In another aspect, the invention concerns a method for the treatment ofa cancer in a human in need thereof comprising administering to suchhuman an effective amount of an anti-HER2 antibody-drug conjugate and aselective Bcl-2 inhibitor.

In one embodiment, the cancer is HER2 positive cancer.

In another embodiment, the cancer is HER2 positive breast cancer orgastric cancer.

In yet another embodiment, the HER2-positive breast cancer or gastriccancer has a HER2 immunohistochemistry (IHC) score of 2+ or 3+ and/or anin situ hybridization (ISH) amplification ratio (her2:CEP17 in situhybridization (ISH) amplification ratio) of 2.0.

In a further embodiment, the HER2-positive cancer, such as breast canceror gastric cancer, is resistant to treatment with an anti-HER2antibody-drug conjugate administered as a single agent.

In a still further embodiment, the HER2-positive cancer, such as breastcancer or gastric cancer, is sensitive to treatment with an anti-HER2antibody-drug conjugate administered as a single agent, and thecombination of the anti-HER2 antibody-drug conjugate and the selectiveBcl-2 inhibitor can be administered to a patient naïve to treatment withthe anti-HER2 antibody-drug conjugate.

In a different embodiment, the anti-HER2 antibody-drug conjugate and theselective Bcl-2 inhibitor show synergistic activity, including, but notlimited to, synergistic activity in a HER2-positive cancer, such asbreast cancer or gastric cancer, that is resistant to treatment with ananti-HER2 antibody-drug conjugate administered as a single agent.

In all embodiments, the anti-HER2 antibody-drug conjugate can, forexample, be trastuzumab-MCC-DM1.

In all embodiments, the selective Bcl-2 inhibitor can, for example, be2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof.

In another aspect, the invention concerns a method for the treatment ofHER2 positive cancer in a human in need thereof comprising administeringto the human an effective amount of trastuzumab-MCC-DM1 and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof.

In one embodiment, the cancer is HER2 positive breast cancer or gastriccancer.

In another embodiment, the HER2-positive breast cancer or gastric cancerhas a HER2 immunohistochemistry (IHC) score of 2+ or 3+ and/or an Insitu hybridization (ISH) amplification ratio (her2:CEP17 in situhybridization (ISH) amplification ratio) of 22.0.

In yet another embodiment, the HER2 positive cancer, such as breastcancer or gastric cancer, is resistant to treatment withtrastuzumab-MCC-DM1 administered as a single agent.

In a further embodiment, the HER2-positive cancer, such as breast canceror gastric cancer, is sensitive to treatment with trastuzumab-MCC-DM1administered as a single agent, and the combination of thetrastuzumab-MCC-DM1 and2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof can be administered to apatient naïve to treatment with trastuzumab-MCC-DM1.

In a still further embodiment, the HER2-positive cancer, such as breastcancer or gastric cancer, is sensitive to treatment with an anti-HER2antibody-drug conjugate administered as a single agent, and thecombination of the anti-HER2 antibody-drug conjugate and the selectiveBcl-2 inhibitor can be administered to a patient naïve to treatment withthe anti-HER2 antibody-drug conjugate. In a further embodiment, thetrastuzumab-MCC-DM1 and the2-1H-pyrrolo[2,3-b]pyridin-5-yloxy)4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptably salt thereof show synergistic activity,including, but not limited to, synergistic activity in a HER2-positivecancer, such as breast cancer or gastric cancer.

In a still further embodiment, the trastuzumab-MCC-DM1 and the2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof are co-administered.

In another embodiment, the trastuzumab-MCC-DM1 and the2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof are administeredsimultaneously.

In yet another embodiment, the trastuzumab-MCC-DMI and the2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof are administeredconsecutively.

In another aspect, the invention concerns the use of a combination of ananti-HER2 antibody-drug conjugate and an inhibitor of a Bcl familyprotein in the preparation of a medicament for the treatment of cancer.

In one embodiment, the Bcl family protein is a Bcl-2 like protein, suchas Mcl-1, Bcl-xl, Bcl-w (BCL2L2), or Bcl-xs, preferably Mcl-1 or Bcl-xl.

In another aspect, the invention concerns the use of a combination of ananti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor in thepreparation of a medicament for the treatment of cancer.

In one embodiment, the invention concerns the use of a combination oftrastuzumab-MCC-DM1 and2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof in the preparation of amedicament for the treatment of cancer.

In all embodiments, the cancer may be HER2 positive cancer.

In all embodiments, the cancer may, for example, be breast cancer orgastric cancer.

In all embodiments, the HER2 positive cancer, such as breast cancer orgastric cancer, may be resistant to treatment with trastuzumab-MCC-DM1administered as a single agent.

In all embodiments, the HER2-positive cancer, such as breast cancer orgastric cancer, may be sensitive to treatment with trastuzumab-MCC-DM1administered as a single agent, and the combination of thetrastuzumab-MCC-DM1 and2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof may be used to treat apatient naïve to treatment with trastuzumab-MCC-DM1.

In another aspect, the invention concerns the use of a combination of ananti-HER2 antibody-drug conjugate and an inhibitor of a Bcl familyprotein in the preparation of a medicament for the treatment of cancer.

In one embodiment, the Bcl family protein is a Bcl-2 like protein, suchas Mcl-1, Bcl-xl, Bcl-w (BCL2L2), or Bcl-xs, preferably Mcl-1 or Bcl-xl.

In a further aspect, the invention concerns a combination of ananti-HER2 antibody-drug conjugate and a selective Bcl-2 inhibitor foruse in the treatment of cancer.

In one embodiment, the combination of trastuzumab-MCC-DM1 and2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof is for use in thetreatment of cancer.

In all combinations, the cancer may, for example, be HER2 positivecancer, such as HER2 positive breast cancer or gastric cancer.

In a particular embodiment, the cancer, such as breast cancer or gastriccancer, is resistant to treatment with the anti-HER2 antibody-drugconjugate or the trastuzumab-MCC-DM1, when administered as a singleagent.

In another embodiment, the HER2-positive cancer, such as breast canceror gastric cancer, may be sensitive to treatment with the anti-HER2antibody-drug conjugate, e.g. trastuzumab-MCC-DM1, when administered asa single agent, and the combination may be used to treat a patient naïveto treatment the anti-HER2 antibody-drug conjugate, e.g.trastuzumab-MCC-DM1.

In another aspect, the invention concerns a method for the diagnosis ofa HER2-positive tumor resistant to treatment with an anti-HER2antibody-drug conjugate, comprising determining in a tumor sampleobtained from a patient with HER2-positive cancer the expression levelof the Bcl-2 gene or its product relative to the expression level in acontrol sample, and diagnosing said cancer as resistant to treatmentwith said anti-HER2 antibody-drug conjugate when the expression level insaid tumor sample is at least 2 fold, or at least 3 fold or at least 4fold, or at least 5 fold greater than the expression level in saidcontrol sample.

In a further aspect, the invention concerns a method for the diagnosisof a HER2-positive tumor susceptible to treatment with an anti-HER2antibody-drug conjugate, comprising determining in a tumor sampleobtained from a patient with HER2-positive cancer the expression levelof the Bcl-2 gene or its product relative to the expression level in acontrol sample, and diagnosing said cancer as susceptible to treatmentwith said anti-HER2 antibody-drug conjugate when the expression level insaid tumor sample is less than 2 fold, or at least 3 fold, or at least 4fold, or at least 5 fold greater than the expression level in saidcontrol sample.

In a still further aspect, the invention concerns a method for thediagnosis of a subject with a HER2-positive tumor as being resistant orsusceptible to treatment with an anti-HER2 antibody-drug conjugate,comprising (i) obtaining a tumor sample from said subject, (ii)measuring the expression level of the Bcl-2 gene or its product in saidtumor sample relative to a control sample, and (iii) diagnosing saidtumor as being resistant to treatment with an anti-HER2 antibody-drugconjugate when the measured expression level of Bcl-2 in said tumorsample is at least 2 fold, or at least 3 fold, or at least 4 fold, or atleast 5 fold greater than the expression level in said control sample,or diagnosing said tumor as being susceptible to treatment with ananti-HER2 antibody-drug conjugate when the measured expression level ofBcl-2 in said tumor sample is less than 2 fold, or less than 3 fold, orless than 4 fold, or less than 5 fold greater than the expression levelin said control sample.

In one embodiment, the subject is a human patient.

In another embodiment, the control sample is a tumor sample of the samecell type that is not resistant to treatment with said anti-HER2antibody-drug conjugate.

In yet another embodiment, the tumor is breast cancer or gastric cancer.

In a further embodiment, the tumor sample is a formalin-fixed,paraffin-embedded tumor sample.

In all embodiments, the diagnostic method may further comprise a step ofmeasuring the expression level of the HER2 gene or its product in thetumor sample.

In all embodiments, the diagnostic method may further comprise a step oftreating the subject with an anti-HER2 antibody-drug conjugate and aselective Bcl-2 inhibitor when the measured expression level of Bcl-2 inthe tumor sample is at least 2 fold, or at least 3 fold, or at least 4fold, or at least 4 fold, or at least 5 fold greater than the expressionlevel in said control sample.

In all embodiments, the diagnostic method may further comprise a step oftreating said patient with an anti-HER2 antibody-drug conjugate when themeasured expression level of Bcl-2 in the tumor sample is less than2-times greater than the expression level in said control sample.

In one embodiment, the anti-HER2 antibody-drug conjugate istrastuzumab-MCC-DM1.

In another embodiment, the selective Bcl-2 inhibitor2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof.

In another aspect, the invention concerns a kit for the in vitrodiagnosis or prognosis of a HER2 positive tumor resistant to treatmentwith an anti-HER2 antibody-drug conjugate in a biological sampleobtained from a patient, which comprises a specific binding partner forthe Bcl-2 gene or its expression product.

In one embodiment, the binding partner is an anti-Bcl-2 antibody.

In another embodiment, the binding partner is a nucleic acid hybridizingto the Bcl-2 gene.

In a further aspect, the invention relates to a kit comprising ananti-HER2 antibody drug conjugate and a selective Bcl-2 inhibitor forthe combination treatment of a patient with a HER2 expressing cancer.

In an embodiment of the present invention, the kit further comprises apharmaceutically acceptable carrier. The kit may further include asterile diluent, which is preferably stored in a separate additionalcontainer. The kit may further Include a package insert comprisingprinted instructions directing the use of the combined treatment as amethod for a HER2 expressing cancer, such as HER2 expressing breast orgastric cancer.

Just as in other aspects, the HER2 expressing cancer may, for example,be breast cancer or gastric cancer, and in various embodiments theanti-HER2 antibody drug conjugate may be trastuzumab-MCC-DM1 and/or theselective Bcl-2 inhibitor may be2-(H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof.

In a particular embodiment, the HER2 expressing cancer to be treated,such as breast cancer or gastric cancer, is resistant to treatment withthe anti-HER2 antibody-drug conjugate or the trastuzumab-MCC-DM1, whenadministered as a single agent.

In another embodiment, the HER2 expressing cancer, such as breast canceror gastric cancer, may be sensitive to treatment with the anti-HER2antibody-drug conjugate, e.g. trastuzumab-MCC-DM1, when administered asa single agent, and the combination may be used to treat a patient naïveto treatment the anti-HER2 antibody-drug conjugate, e.g.trastuzumab-MCC-DM1.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show expression of Bcl-2 family pro-survival moleculesin T-DM1 resistant KPL-4 and BT-474M1 human breast cancer cells (HER2positive) relative to parental cells. FIG. 1A shows mRNA expressionassessed by TaqMan qRT-PCR analysis; FIG. 1B shows protein expression byWestern blot analysis.

FIG. 2 presents results of a cell viability assay showing that theT-DM1+GDC-0199 combination has a synergistic effect in KPL-4T-DM1-resistant human breast cancer cells, while no synergism isobserved in KPL-4 parental cells.

FIG. 3 presents results of a caspase activation assay measuringactivation of caspases 3 and 7. The results show that at 24 hours ofdrug treatment, T-DM1-resistant KPL-4 human breast cancer cells arere-sensitized to T-DM1 in the presence of GDC-0199, whereas at 24 hours,there is no effect of T-DM1+/−GDC-0199 in KPL-4 parental cells.

FIGS. 4A and 4B presents the results of a caspase activation assaymeasuring activation of caspases 3 and 7 in T-DM1 resistant KPL-4 humanbreast cancer cells relative to parental cells after 48 hours of drugtreatment. The results show dose-dependent increases in caspases 3 and 7activation with the addition of GDC-0199 to T-DM1, with minimal effectof T-DM1 alone. In KPL-4 parental cells, T-DM1 induces robust activationof caspases 3 and 7 with minimal increase upon addition of GDC-0199.

FIG. 5A presents the results of a caspase activation assay measuringactivation of caspases 3 and 7 in Clone #17 T-DM1 resistant KPL-4 humanbreast cancer cell line treated with 1 ug/mL T-DM1 alone or incombination with the indicated doses of GDC-0199 for 48 hours. Theresults show dose-dependent increases in caspases 3 and 7 activationwith the addition of GDC-0199 to T-DM1, with minimal effect of T-DM1alone.

FIG. 5B shows the effect of T-DM1 (5 mg/kg administered once), GDC-0199(100 mg/kg qd×21) or the combination on tumor growth (as measured bytumor volume) of Clone #17 T-DM1 resistant KPL-4 human breast cancerxenografts in SCID beige mice. Combination drug treatment resulted intumor stasis, with no activity of single agent treatment.

FIGS. 6A and 6B show the results of a caspase activation assay measuringactivation of caspases 3 and 7 in Clone #8 T-DM1 resistant KPL-4 humanbreast cell line at 0.1 pg/mL and 1pg/mL T-DM1 concentrations,respectively, alone or in combination with the indicated concentrationsof GDC-199.

FIG. 6C shows the effect of T-DM1 (5 mg/kg q3w×2), GDC-0199 (100 mg/kgqd×21) or the combination on tumor growth (as measured by tumor volume)of Clone #8 T-DM1 resistant KPL-4 human breast cancer xenografts in SCIDbeige mice.

FIG. 7A shows the expression of Bcl-2 in formalin-fixedparaffin-embedded T-DM1 resistant KPL-4 xenograft tumor samples (Clones#8 and #17) determined by immunohistochemistry (IHC), using DABdetection method.

FIG. 7B shows the expression of HER2 (ErbB2) in formalin-fixedparaffin-embedded T-DM1 resistant KPL-4 xenograft tumor samples (Clones#8 and #17) determined by immunohistochemistry (IHC), using DABdetection method.

FIG. 8 shows protein expression as measured by Western blot analysis ofBcl-2 and HER2 in Clone #17 KPL-4 T-DM1-resistant xenograft tumors,treated with GDC-0199, T-DM1 or T-DM-1+GDC-0199.

FIG. 9A presents results of a caspase 3/7 activation luminescent invitro apoptosis assay, testing the effect of five separateconcentrations of GDC-0199 (μM) in combination with 9 differentconcentrations of T-DM1 after 24 hours of treatment in HER2+MDA-MB-361breast cancer cells (T-DM1 naïve cells). The results demonstrateenhanced apoptosis greater than T-DM1 alone with all combinationstested.

FIG. 9B presents the results of a kinetic caspase 3/7 activationfluorescent in vitro apoptosis assay, testing the effect of threedifferent concentrations of GDC-0199 (0.63 μM, 1.25 μM, 2.5 LM), aloneand in combination with T-DM1 (0.1 μg/mL), in HER2+MDA-MB-361 breastcancer cells. The results demonstrate enhanced caspase activationgreater than T-DM1 alone with all combinations tested.

FIG. 10A presents the results of a caspase 3/7activation luminescent invitro apoptosis assay, testing the effect of five separateconcentrations of GDC-0199 (μM) in combination with 9 differentconcentrations for 24 hours of treatment in HER2+HCC1569 breast cancercells (T-DM1 naïve cells). The results demonstrate no induction ofapoptosis by T-DM1 alone but enhanced apoptosis with all combinationstested.

FIG. 10B presents the results of a kinetic caspase 3/7 fluorescent invitro apoptosis assay, testing the effect of three differentconcentrations of GDC-0199 (0.63 μM, 1.25 μM, 2.5 μM), alone and incombination with T-DM1 (0.1 μg/mL), in HER2+HCC1569 breast cancer cells.The results demonstrate dose-dependent enhanced caspase activation withcombination treatment.

FIG. 11 shows the effect of T-DM1 (1 mg/kg, 3 mg/kg, 7 mg/kg, iv q3w×1)and GDC-0199 (100 mg/kg, po, qd×21), alone or in combination, on tumorgrowth in HER2+MDA-MB-361 breast cancer xenograft model. Significanttumor growth delay was observed with GDC-0199 combined with 7 mg/kgT-DM1.

FIG. 12 shows Western blot analysis of the effects of T-DM1, with orwithout GDC-0199, on Bcl-2 family members (total and phospho-Bcl-2 and-Bcl-xL, total Mcl-1 and Bim) in HER2+breast cancer cell lines BT-474,EFM192A, KPL-4, HCC1569, HCC1954, MDA-361, UACC-812, ZR-75-30.

FIG. 13 shows the amino acid sequence of trastuzumab light chain (SEQ IDNO: 1).

FIG. 14 shows the amino acid sequence of trastuzumab heavy chain (SEQ IDNO: 2).

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described.

All references cited throughout the disclosure are expresslyincorporated by reference herein in their entirety. In the event thatone or more of the incorporated literature, patents, and similarmaterials differs from or contradicts this application, including butnot limited to defined terms, term usage, described techniques, or thelike, this application controls.

Definitions

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The anti-HER2 antibody used in the antibody-drug conjugates herein is amonoclonal antibody.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred. Such murine/human chimeric antibodiesare the product of expressed immunoglobulin genes comprising DNAsegments encoding murine immunoglobulin variable regions and DNAsegments encoding human immunoglobulin constant regions. Other forms of“chimeric antibodies” encompassed by the present invention are those inwhich the class or subclass has been modified or changed from that ofthe original antibody. Such “chimeric” antibodies are also referred toas “class-switched antibodies.” Methods for producing chimericantibodies involve conventional recombinant DNA and gene transfectiontechniques now well known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR of an immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a murine CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody.” See, e.g.,Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S.,et al., Nature 314 (1985) 268-270. Particularly preferred CDRscorrespond to those representing sequences recognizing the antigensnoted above for chimeric and bifunctional antibodies.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. Human antibodies are well-known inthe state of the art (van Disk, M. A., and van de Winkel, J. G., Curr.Opin. Pharmacol. 5 (2001) 368-374) and can be produced by a variety oftechniques, including phage display. Based on such technology, humanantibodies against a great variety of targets can be produced. Examplesof human antibodies are for example described in Kellermann, S. A., etal., Curr Opin Biotechnol. 13 (2002) 593-597. For the use of phagedisplay technology to produce and select human antibodies see, e.g.,Winter et al., Ann Review Immunol, 1994, 12:433-455; and for theproduction of fully human antibodies from transgenic mouse and phagedisplay platforms see, e.g., Lonberg, Current Opinion Immunol, 2008,20(4):450-459.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as, for example, antibodies isolatedfrom a host cell such as a NS0 or CHO cell or from an animal (e.g. amouse) that is transgenic for human immunoglobulin genes or antibodiesexpressed using a recombinant expression vector transfected into a hostcell. Such recombinant human antibodies have variable and constantregions derived from human germline immunoglobulin sequences in arearranged form.

As used herein, “specifically binding” or “binds specifically to” refersa binding that is sufficiently selective to a target as to distinguishit from a binding to unwanted or nonspecific targets. In one embodiment,an antibody that binds specifically to a target will have a bindingaffinity for that target (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³M). In yet another embodiment, the KD is10⁻¹⁰ mol/l or lower (e.g. 10⁻¹² mol/l). The binding affinity isdetermined with a standard binding assay, such as Scatchard plotanalysis on cells expressing the target antigen.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded. In one embodiment, it isdouble-stranded DNA.

The “constant domains” are not involved directly in binding the antibodyto an antigen but are involved in the effector functions (ADCC,complement binding, and CDC).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Frccman and Co., page 91(2007).)

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

The term “anti-HER2 antibody” according to the invention is an antibodythat binds specifically to HER2 antigen.

As defined herein, the terms “trastuzumab”, “HERCEPTIN®” and“huMAb4D5-8” are used interchangeably. Such antibody preferablycomprises the light and heavy chain amino acid sequences shown in FIG.13 (SEQ ID NO: 1) and FIG. 14 (SEQ ID NO. 2), respectively.

The “epitope 4D5” or “4D5 epitope” or “4D5” is the region in theextracellular domain of HER2 to which the antibody 4D5 (ATCC CRL 10463)and trastuzumab bind. This epitope is close to the transmembrane domainof HER2, and within Domain IV of HER2. To screen for antibodies whichbind to the 4D5 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 4D5 epitope of HER2 (e.g. any one or more residuesin the region from about residue 529 to about residue 625, inclusive, ofHER2).

The “epitope 2C4” or “2C4 epitope” is the region in the extracellulardomain of HER2 to which the antibody 2C4 binds. In order to screen forantibodies which bind to the 2C4 epitope, a routine cross-blocking assaysuch as that described in Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping can be performed to assess whether theantibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprisesresidues from domain II in the extracellular domain of HER2. The 2C4antibody and Pertuzumab bind to the extracellular domain of HER2 at thejunction of domains I, II and III (Franklin et al. Cancer Cell 5:317-328(2004)).

As defined herein, the terms “T-DM1,” “trastuzumab-MCC-DM1,”“ado-trastuzumab emtansine,” “trastuzumab emtansine,” and “KADCYLA” areused interchangeably, and refer to trastuzumab linked through the linkermoiety MCC to the maytansinoid drug moiety DM1, including all mixturesof variously loaded and attached antibody-drug conjugates where 1, 2, 3,4, 5, 6, 7, and 8 drug moieties are covalently attached to the antibodytrastuzumab (U.S. Pat. No. 7,097,840; US 2005/0276812; US 2005/0166993).

The term “Bcl-2” as used herein refers to the Bcl-2 protein (Swiss ProtID No. P10415), a member of the Bcl-2 family of proteins (Cory, S., andAdams, J. M., Nature Reviews Cancer 2 (2002) 647-656; Adams, Genes undDevelopment 17 (2003) 2481-2495; Danial, N. N., and Korsmeyer, S. J.,Cell 116 (2004) 205-219; Petros, A. M., Biochim Biophys Acta 1644 (2004)83-94).

The term “selective Bcl-2 inhibitor” as used herein refers topolypeptides and small molecules inhibiting prosurvival members of theBcl-2 family of proteins. Preferably, the selective Bcl-2 inhibitor is2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide.(a.k.a. ABT-199 or GDC-0199), or a pharmaceutically acceptable saltthereof, a Bcl-2 inhibitor of formula I, which is described inInternational Publication No. WO2010/0138588 and in US publication No.US2010/0305122, which are incorporated by reference herein.

Herein, an “anti-tumor agent” refers to a drug used to treat cancer.Non-limiting examples of anti-tumor agents herein include chemotherapyagents, HER dimerization inhibitors, HER antibodies, antibodies directedagainst tumor associated antigens, anti-hormonal compounds, cytokines,EGFR-targeted drugs, anti-angiogenic agents, tyrosine kinase inhibitors,growth inhibitory agents and antibodies, cytotoxic agents, antibodiesthat induce apoptosis, COX inhibitors, farnesyl transferase inhibitors,antibodies that binds oncofetal protein CA 125, HER2 vaccines, Raf orras inhibitors, liposomal doxorubicin, topotecan, taxane, dual tyrosinekinase inhibitors, TLK286, EMD-7200, pertuzumab, trastuzumab, erlotinib,and bevacizumab.

A “chemotherapy” is use of a chemotherapeutic agent useful in thetreatment of cancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Examples of chemotherapeuticagents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel(TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No.51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9,Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No.15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®,Bristol-Myers Squibb Oncology, Princeton, N.J.), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifamib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GaxoSmithKline), canfosfamide (TELCYTA, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlomaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate, hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A,roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C);cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine(NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptably salts,acids and derivatives of any of the above.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include breast cancer, squamous cellcancer (e.g., epithelial squamous cell cancer), lung cancer includingsmall-cell lung cancer, non-small cell lung cancer (“NSCLC”),adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer. In apreferred embodiment, the cancer is breast cancer. In another preferredembodiment, the cancer is gastric cancer.

Reference to a tumor or cancer as a “Stage 0,” “Stage I,” “Stage II,”“Stage III,” or “Stage IV”, and various sub-stages within thisclassification, indicates classification of the tumor or cancer usingthe Overall Stage Grouping or Roman Numeral Staging methods known in theart. Although the actual stage of the cancer is dependent on the type ofcancer, in general, a Stage 0 cancer is an in situ lesion, a Stage Icancer is small localized tumor, a Stage II and III cancer is a localadvanced tumor which exhibits involvement of the local lymph nodes, anda Stage IV cancer represents metastatic cancer. The specific stages foreach type of tumor is known to the skilled clinician.

The term “metastatic breast cancer” means the state of breast cancerwhere the cancer cells are transmitted from the original site to one ormore sites elsewhere in the body, by the blood vessels or lymphatics, toform one or more secondary tumors in one or more organs besides thebreast.

An “advanced” cancer is one which has spread outside the site or organof origin, either by local invasion or metastasis. Accordingly, the term“advanced” cancer includes both locally advanced and metastatic disease.

A “refractory” cancer is one which progresses even though an anti-tumoragent, such as a chemotherapy, is being administered to the cancerpatient. An example of a refractory cancer is one which is platinumrefractory.

A “recurrent” cancer is one which has regrown, either at the initialsite or at a distant site, after a response to initial therapy, such assurgery.

A “locally recurrent” cancer is cancer that returns after treatment inthe same place as a previously treated cancer.

An“operable” or“resectable” cancer is cancer which is confined to theprimary organ and suitable for surgery (resection).

A “non-resectable” or “unresectable” cancer is not able to be removed(resected) by surgery.

The terms “HER2-positive” and “HER2 expressing” are used hereininterchangeably. A “HER2-positive” cancer comprises cancer cells whichhave higher than normal levels of HER2. Examples of HER2-positive cancerinclude HER2-positive breast cancer and HER2-positive gastric cancer.Optionally, HER2-positive is a HER2 overexpressing cancer, and incertain embodiments the HER-2 positive cancer has animmunohistochemistry (IHC) score of 2+ or 3+ and/or an in situhybridization (ISH) amplification ratio ≥2.0.

In situ hybridization (ISH) determines the number of her2 copies using aDNA probe coupled to a fluorescent, chromogenic, or silver detectionsystem (ie, FISH, CISH, or SISH), or a combination of CISH and SISHsystems (bright-field double ISH (BDISH) or dual-hapten, dual-color ISH(DDISH)). ISH may be conducted using a single probe to enumerate her2copies per nucleus only or as a dual-probe technique where hybridizationof a chromosome 17 centromere probe (chromosome enumeration probe 17,CEP17) allows determination of the her2:CEP17 ratio. The two-probeapproach may be performed as a dual-color technique, withcohybridization of the two probes on the same slide, or as a monochromeassay where each probe is used on sequential slides. The her2:CEP17ratio is sometimes regarded as a better reflection of her2 amplificationstatus than mean her2 copy number, as the latter is also dependent onother parameters, such as the mitotic index of the tumor, sectionthickness, nuclear truncation effects, and abnormal chromosome copynumber (aneusomy). The phrase “in situ hybridization (ISH) amplificationratio ≥2.0” refers to her2:CEP17 ratio ≥2.0. For further details see,e.g. Sauter G, et al. Guidelines for human epidermal growth factorreceptor 2 testing: biologic and methodologic considerations. J ClinOncol 2009; 27:1323-1333, and the review article by Hanna et al. ModernPathology (2014) 27, 4-18.

Herein, a “patient” or “subject” is a human patient. The patient may bea “cancer patient,” i.e. one who is suffering or at risk for sufferingfrom one or more symptoms of cancer, in particular gastric or breastcancer.

A “patient population” refers to a group of cancer patients. Suchpopulations can be used to demonstrate statistically significantefficacy and/or safety of a drug, such as Pertuzumab.

A “relapsed” patient is one who has signs or symptoms of cancer afterremission. Optionally, the patient has relapsed after adjuvant orneoadjuvant therapy.

A cancer or biological sample which “displays HER expression,amplification, or activation” is one which, in a diagnostic test,expresses (including overexpresses) a HER receptor, has amplified HERgene, and/or otherwise demonstrates activation or phosphorylation of aHER receptor.

The term “synergistic” as used herein refers to a therapeuticcombination which is more effective than the additive effects of the twoor more single agents. A determination of a synergistic interactionbetween an anti-Her2 antibody-dug conjugate, such astrastuzumab-MCC-DM1, and a selective Bcl-2 inhibitor may be based on theresults obtained from the assays described herein, or in other assaysystems known in the art, utilizing a standard programs for quantifyingsynergism, additivism, and antagonism among anticancer agents. Theprogram preferably utilized is that described by Chou and Talalay, in“New Avenues in Developmental Cancer Chemotherapy,” Academic Press,1987, Chapter 2. Combination Index (CI) values less than 0.8 indicatesynergy, values greater than 1.2 indicate antagonism and values between0.8 to 1.2 indicate additive effects. The combination therapy mayprovide “synergy” and prove “synergistic”, i.e., the effect achievedwhen the active ingredients used together is greater than the sum of theeffects that results from using the compounds separately. A synergisticeffect may be attained when the active ingredients are: (1)co-formulated and administered or delivered simultaneously in acombined, unit dosage formulation; (2) delivered by alternation or inparallel as separate formulations; or (3) by some other regimen. Whendelivered in alternation therapy, a synergistic effect may be attainedwhen the compounds are administered or delivered sequentially, e.g., bydifferent injections in separate syringes. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially in time, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

“Neoadjuvant therapy” or“preoperative therapy” herein refers to therapygiven prior to surgery.

The goal of neoadjuvant therapy is to provide immediate systemictreatment, potentially eradicating micrometastases that would otherwiseproliferate if the standard sequence of surgery followed by systemictherapy were followed. Neoadjuvant therapy may also help to reduce tumorsize thereby allowing complete resection of initially unresectabletumors or preserving portions of the organ and its functions.Furthermore, neoadjuvant therapy permits an in vivo assessment of drugefficacy, which may guide the choice of subsequent treatments.

“Adjuvant therapy” herein refers to therapy given after definitivesurgery, where no evidence of residual disease can be detected, so as toreduce the risk of disease recurrence. The goal of adjuvant therapy isto prevent recurrence of the cancer, and therefore to reduce the chanceof cancer-related death. Adjuvant therapy herein specifically excludesneoadjuvant therapy.

“Definitive surgery” is used as that term is used within the medicalcommunity. Definitive surgery includes, for example, procedures,surgical or otherwise, that result in removal or resection of the tumor,including those that result in the removal or resection of all grosslyvisible tumor. Definitive surgery includes, for example, complete orcurative resection or complete gross resection of the tumor. Definitivesurgery includes procedures that occur in one or more stages, andincludes, for example, multi-stage surgical procedures where one or moresurgical or other procedures are performed prior to resection of thetumor. Definitive surgery includes procedures to remove or resect thetumor including involved organs, parts of organs and tissues, as well assurrounding organs, such as lymph nodes, parts of organs, or tissues.Removal may be incomplete such that tumor cells might remain even thoughundetected.

“Survival” refers to the patient remaining alive, and includes diseasefree survival (DFS), progression free survival (PFS) and overallsurvival (OS). Survival can be estimated by the Kaplan-Meier method, andany differences in survival are computed using the stratified log-ranktest.

“Progression-Free Survival” (PFS) is the time from the first day oftreatment to documented disease progression (including isolated CNSprogression) or death from any cause on study, whichever occurs first.

“Disease free survival (DFS)” refers to the patient remaining alive,without return of the cancer, for a defined period of time such as about1 year, about 2 years, about 3 years, about 4 years, about 5 years,about 10 years, etc., from initiation of treatment or from initialdiagnosis. In one aspect of the invention, DFS is analyzed according tothe intent-to-treat principle, i.e., patients are evaluated on the basisof their assigned therapy. The events used in the analysis of DFS caninclude local, regional and distant recurrence of cancer, occurrence ofsecondary cancer, and death from any cause in patients without a priorevent (e.g, breast cancer recurrence or second primary cancer).

“Overall survival” refers to the patient remaining alive for a definedperiod of time, such as about 1 year, about 2 years, about 3 years,about 4 years, about 5 years, about 10 years, etc., from initiation oftreatment or from initial diagnosis. In the studies underlying theinvention the event used for survival analysis was death from any cause.

By “extending survival” is meant increasing DFS and/or OS in a treatedpatient relative to an untreated patient, or relative to a controltreatment protocol. Survival is monitored for at least about six months,or at least about 1 year, or at least about 2 years, or at least about 3years, or at least about 4 years, or at least about 5 years, or at leastabout 10 years, etc., following the initiation of treatment or followingthe initial diagnosis.

“Hazard ratio” in survival analysis is a summary of the differencebetween two survival curves, representing the reduction in the risk ofdeath on treatment compared to control, over a period of follow-up.Hazard ratio is a statistical definition for rates of events. For thepurpose of the invention, hazard ratio is defined as representing theprobability of an event in the experimental arm divided by theprobability of an event in the control arm at any specific point intime.

By “monotherapy” is meant a therapeutic regimen that includes only asingle therapeutic agent for the treatment of the cancer or tumor duringthe course of the treatment period.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the growth, development or spread of ahyperproliferative condition, such as cancer. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

The term “a method of treating” or its equivalent, when applied to, forexample, cancer refers to a procedure or course of action that isdesigned to reduce or eliminate the number of cancer cells in a patient,or to alleviate the symptoms of a cancer. “A method of treating” canceror another proliferative disorder does not necessarily mean that thecancer cells or other disorder will, in fact, be eliminated, that thenumber of cells or disorder will, in fact, be reduced, or that thesymptoms of a cancer or other disorder will, in fact, be alleviated.Often, a method of treating cancer will be performed even with a lowlikelihood of success, but which, given the medical history andestimated survival expectancy of a patient, is nevertheless deemed toinduce an overall beneficial course of action.

The terms “co-administration” or “co-administering” refer to theadministration of the anti-HER2 antibody-drug conjugate and theselective Bcl-2 inhibitor as two separate formulations. Theco-administration can be simultaneous or sequential in cither order. Inone further embodiment, there is a time period while both (or all)active agents simultaneously exert their biological activities. Theanti-HER2 antibody-drug conjugate and the selective Bcl-2 inhibitor areco-administered either simultaneously or sequentially (e.g. via anintravenous (i.v.) through a continuous infusion (one for theantibody-drug conjugate and eventually one for the Bcl-2 inhibitor; orthe Bcl-2 inhibitor is administered orally). When both therapeuticagents are co-administered sequentially the agents are administered intwo separate administrations that are separated by a “specific period oftime”. The term specific period of time is meant anywhere from 1 hour to15 days. For example, one of the agents can be administered within about15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, or 24, 23, 22,21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2or 1 hour from the administration of the other agent, and, in oneembodiment, the specific period time is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1day, or 24, 23, 22, 21, 20, 19, 18,17,16,15,14,13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1 hour.

The term “simultaneously” means at the same time or within a shortperiod of time, usually less than 1 hour.

A drug that is administered “concurrently” with one or more other drugsis administered during the same treatment cycle, on the same day oftreatment as the one or more other drugs, and, optionally, at the sametime as the one or more other drugs. For instance, for cancer therapiesgiven every 3 weeks, the concurrently administered drugs are eachadministered on day-1 of a 3-week cycle.

A dosing period as used herein is meant a period of time, during whicheach therapeutic agent has been administered at least once. A dosingcycle is usually about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

In certain embodiments, a dosing cycle is for 21 days.

In certain embodiments of a method of treating cancer in a patient asprovided herein, the method comprises administering the anti-HER2antibody-drug conjugate and the selective Bcl-2 inhibitor for one ormore dosing cycles to the patient. In one embodiment, the one or moredosing cycles each last for at least one week. In another embodiment,the one or more dosing cycles are each for at least two weeks, threeweeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nineweeks, or for more than nine weeks. In one embodiment, each dosing cycleis three weeks.

In a preferred embodiment, the antibody-drug conjugate is administeredas an intravenous (i.v.) infusion every three weeks (21-day cycle).

In another preferred embodiment, the antibody-drug conjugate is KADCYLA®(ado-trastuzumab emtansine), which is administered as a 3.6 mg/kg i.v.infusion every 3 weeks (21-day cycle).

In some embodiments of the method of treatment provided herein, theselective Bcl-2 inhibitor is2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide(GDC-0199). In certain embodiments, the amount of GDC-0199 administeredto the patient per dose is increased during the first dosing cycle frominitial amounts of between 10 mg to 80 mg to final amounts of between190 mg to 400 mg. In certain embodiments, the amount of GDC-0199 perdose administered to the patients begins with 50 mg or 100 mg, and isincreased to 300 mg per dose. In some embodiments, the amount ofGDC-0199 in the initial doses administered to the patient can, forexample, be between 20 mg to 60 mg (e.g., 20 mg, 25 mg, 30 mg, 35 mg, 40mg, 45 mg, 50 mg, 55 mg or 60 mg doses), followed by dose amounts of 100mg, 200 mg, 300 mg or more of GDC-0199.

An “adverse event” is any unfavorable and unintended sign, symptom, ordisease temporally associated with the use of an investigational(medicinal) product or other protocol-imposed intervention, regardlessof attribution; and includes: adverse events (AEs) not previouslyobserved in the patient that emerge during the protocol-specified AEreporting period, including signs or symptoms associated with breastcancer that were not present before the AE reporting period;complications that occur as a result of protocol-mandated interventions(e.g., invasive procedures such as biopsies); if applicable, AEs thatoccur before assignment of study treatment associated with medicationwashout, no treatment run-in, or other protocol-mandated intervention;Preexisting medical conditions (other than the condition being studied)judged by the investigator to have worsened in severity or frequency orchanged in character during the protocol-specified AE reporting period.

It is self-evident that the antibody-drug conjugates are administered tothe patient in a “therapeutically effective amount” (or simply“effective amount”) which is the amount of the respective compound orcombination that will elicit the biological or medical response of atissue, system, animal or human that is being sought by the researcher,veterinarian, medical doctor or other clinician. The administration ofan effective amount of a therapeutically agent can be a singleadministration or split dose administration. “split dose administration”is meant an effective amount is a split into multiple doses, preferably2, and administered within 1 or 2 days. For example, if 100 mg of aselective Bcl-2 inhibitor is deemed effective, it can be administered inone 100 mg administration or two 50 mg administrations. Split doseadministration is sometimes desirable at the beginning of a dosingperiod to reduce side effects. When an effective amount is administeredin split dosing, it is still considered one administration of aneffective amount. For example, when 100 mg is the effective amount of aselective Bcl-2 inhibitor and that amount is administered in two 50 mgdoses over a period of time, e.g. 2 days, only one effective amount isadministered during that period of time.

A “fixed” or “flat” dose of a therapeutic agent herein refers to a dosethat is administered to a human patient without regard for the weight(WT) or body surface area (BSA) of the patient. The fixed or flat doseis therefore not provided as a mg/kg dose or a mg/m2 dose, but rather asan absolute amount of the therapeutic agent.

A “loading” dose herein generally comprises an initial dose of atherapeutic agent administered to a patient, and is followed by one ormore maintenance dose(s) thereof. Generally, a single loading dose isadministered, but multiple loading doses are contemplated herein.Usually, the amount of loading dose(s) administered exceeds the amountof the maintenance dose(s) administered and/or the loading dose(s) areadministered more frequently than the maintenance dose(s), so as toachieve the desired steady-state concentration of the therapeutic agentearlier than can be achieved with the maintenance dose(s).

A “maintenance” dose herein refers to one or more doses of a therapeuticagent administered to the patient over a treatment period. Usually, themaintenance doses are administered at spaced treatment intervals, suchas approximately every week, approximately every 2 weeks, approximatelyevery 3 weeks, or approximately every 4 weeks, preferably every 3 weeks.

“Infusion” or “infusing” refers to the introduction of a drug-containingsolution into the body through a vein for therapeutic purposes.Generally, this is achieved via an intravenous (IV) bag.

An “intravenous bag” or “IV bag” is a bag that can hold a solution whichcan be administered via the vein of a patient. In one embodiment, thesolution is a saline solution (e.g. about 0.9% or about 0.45% NaCl).Optionally, the IV bag is formed from polyolefin or polyvinyl chloride.

In the context of this invention, additional other cytotoxic,chemotherapeutic or anti-cancer agents, or compounds that enhance theeffects of such agents (e.g. cytokines) may be used in the anti-HER2antibody-drug conjugate and Bcl-2 inhibitor combination treatment ofHER2 expressing cancer. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.Preferably the anti-HER2 antibody-drug conjugate and Bcl-2 inhibitorcombination treatment is used without such additional cytotoxic,chemotherapeutic or anti-cancer agents, or compounds that enhance theeffects of such agents.

Such agents include, for example: alkylating agents or agents with analkylating action, such as cyclophosphamidc (CTX; e.g. CYTOXAN®),chlorambucil (CHL; e.g. LEUKERAN®), cisplatin (CisP; e.g. PLATINOL®)busulfan (e.g. MYLERAN®), melphalan, carmustine (BCNU), streptozotocin,triethylenemelamine (TEM), mitomycin C, and the like; anti-metabolites,such as methotrexate (MTX), etoposide (VP16; e.g. VEPESID®),6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C),5-fluorouracil (5-FU), capecitabine (e.g. XELODA®), dacarbazine (DTIC),and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g.ADRIAMYCIN®), daunorubicin (daunomycin), bleomycin, mithramycin and thelike; alkaloids, such as vinca alkaloids such as vincristine (VCR),vinblastine, and the like; and other antitumor agents, such aspaclitaxel (e.g. TAXOL®) and paclitaxel derivatives, the cytostaticagents, glucocorticoids such as dexamethasone (DEX; e.g. DECADRON®) andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such ashydroxyurea, amino acid depleting enzymes such as asparaginase,leucovorin and other folic acid derivatives, and similar, diverseantitumor agents.

The following agents may also be used as additional agents: arnifostine(e.g. ETHYOL®), dactinomycin, mechlorethamine (nitrogen mustard),streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g.DOXIL®), gemcitabine (e.g. GEMZAR®), daunorubicin lipo (e.g.DAUNOXOME®), procarbazine, mitomycin, docetaxel (e.g. TAXOTERE®),aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11(irinotecan), 10-hydroxy 7-ethyl-camptothecin (SN38), floxuridine,fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferonalpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan,mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin,pipobroman, plicamycin, tamoxifen, teniposide, testolactone,thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil.Preferably the type II anti-CD20 antibody and Bcl-2 inhibitorcombination treatment is used without such additional agents.

The use of the cytotoxic and anticancer agents described above as wellas antiproliferative target-specific anticancer drugs like proteinkinase inhibitors in chemotherapeutic regimens is generally wellcharacterized in the cancer therapy arts, and their use herein fallsunder the same considerations for monitoring tolerance and effectivenessand for controlling administration routes and dosages, with someadjustments. For example, the actual dosages of the cytotoxic agents mayvary depending upon the patient's cultured cell response determined byusing histoculture methods. Generally, the dosage will be reducedcompared to the amount used in the absence of additional other agents.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

In the context of this invention, an effective amount of ionizingradiation may be carried out and/or a radiopharmaceutical may be used inaddition to the anti-HER2 antibody-drug conjugate and Bcl-2 inhibitorcombination treatment. The source of radiation can be either external orinternal to the patient being treated. When the source is external tothe patient, the therapy is known as external beam radiation therapy(EBRT). When the source of radiation is internal to the patient, thetreatment is called brachytherapy (BT). Radioactive atoms for use in thecontext of this invention can be selected from the group including, butnot limited to, radium, cesium-137, iridium-192, americium-241,gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131,and indium-111. Is also possible to label the antibody with suchradioactive isotopes. Preferably the type II anti-CD20 antibody andBcl-2 inhibitor combination treatment is used without such ionizingradiation.

Radiation therapy is a standard treatment for controlling unresectableor inoperable tumors and/or tumor metastases. Improved results have beenseen when radiation therapy has been combined with chemotherapy.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproductivecells in both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (Gy), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsiderations, but the two most important are the location of the tumorin relation to other critical structures or organs of the body, and theextent to which the tumor has spread. A typical course of treatment fora patient undergoing radiation therapy will be a treatment schedule overa 1 to 6 week period, with a total dose of between 10 and 80 Gyadministered to the patient in a single daily fraction of about 1.8 to2.0 Gy, 5 days a week. In a preferred embodiment of this invention thereis synergy when tumors in human patients are treated with thecombination treatment of the invention and radiation. In other words,the inhibition of tumor growth by means of the agents comprising thecombination of the invention is enhanced when combined with radiation,optionally with additional chemotherapeutic or anticancer agents.Parameters of adjuvant radiation therapies are, for example, containedin WO 99/60023.

The anti-HER2 antibody-drug conjugates are administered to a patientaccording to known methods, by intravenous administration as a bolus orby continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, or intrathecal routes. Intravenous or subcutaneousadministration of the antibodies is preferred.

The Bcl-2 inhibitors are administered to a patient according to knownmethods, e.g. by intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, or peroral routes. Intravenous, subcutaneous or oraladministration of the Bcl-2 inhibitors is preferred.

As used herein, a “pharmaceutically acceptable carrier” is intended toinclude any and all material compatible with pharmaceuticaladministration including solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and other materials and compounds compatible with pharmaceuticaladministration. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsof the invention is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A “vial” is a container suitable for holding a liquid or lyophilizedpreparation. In one embodiment, the vial is a single-use vial, e.g. a20-cc single-use vial with a stopper.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

Trastuzumab-MCC-DM1 (T-DM1: KADCYLA®, ado-trastuzumab emtansine)

The present invention includes therapeutic combinations comprisingtrastuzumab-MCC-DM1 which has the structure:

where Tr is trastuzumab, and p is an integer from 1 to 8. The drug toantibody ratio or drug loading is represented by p in the abovestructure of trastuzumab-MCC-DM1. The drug loading value p is 1 to 8.Trastuzumab-MCC-DM1 includes all mixtures of variously loaded andattached antibody-drug conjugates where 1, 2, 3, 4, 5, 6, 7, and 8 drugmoieties are covalently attached to the antibody trastuzumab.

Trastuzumab is produced by a mammalian cell (Chinese Hamster Ovary, CHO)suspension culture. The HER2 (or c-erbB2) proto-oncogene encodes atransmembrane receptor protein of 185 kDa, which is structurally relatedto the epidermal growth factor receptor. HER2 protein overexpression isobserved in 25%30% of primary breast cancers and can be determined usingan immunohistochemistry based assessment of fixed tumor blocks (Press MF, et al (1993) Cancer Res 53:4960-70. Trastuzumab is an antibody thathas antigen binding residues of, or derived from, the murine 4D5antibody (ATCC CRL 10463, deposited with American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 under theBudapest Treaty on May 24, 1990). Exemplary humanized 4D5 antibodiesinclude huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5,huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®) as in U.S. Pat. No.5,821,337.

The antibody-drug conjugate, trastuzumab-MCC-DM1, comprises amaytansinoid drug moiety DM1 (U.S. Pat. Nos. 5,208,020; 6,441,163) andmay be prepared from ansamitocin fermentation products (U.S. Pat. No.6,790,954; US 2005/0170475).

Selective Bcl-2 Inhibitors

In a preferred embodiment, the selective Bcl-2 inhibitor of the presentinvention is2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl)-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamide.(a.k.a. ABT-199 or GDC-O 199), a Bcl-2 inhibitor of formula I, which isdescribed in International Publication No. WO2010/0138588 and in USpublication NO. US2010/0305122, which are incorporated by referenceherein.

Other selective Bcl-2 inhibitors include, for example, Oblimersen,SPC-2996, RTA-402, Gossypol, AT-101, Obatoclax mesylate, A-371191,A-385358, A-438744, ABT-737, ABT-263, AT-101, BL-11, BL-193, GX-15003,2-Methoxyanitimycin A₃, HA-14-1, KF-67544, Purpurogallin, TP-TW-37,YC-137 and Z-24, described e.g. in Zhai, D., et al., Cell Death andDifferentiation 13 (2006) 1419-1421.

Pharmaceutical Compositions

Pharmaceutical compositions or formulations of the present inventioninclude combinations of trastuzumab-MCC-DM1, a selective Bcl-2inhibitor, and one or more pharmaceutically acceptable carrier, glidant,diluent, or excipient.

Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors of the presentinvention may exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike, and it is intended that the invention embrace both solvated andunsolvated forms.

Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors of the presentinvention may also exist in different tautomeric forms, and all suchforms are embraced within the scope of the invention. The term“tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Pharmaceutical compositions encompass both the bulk composition andindividual dosage units comprised of more than one (e.g., two)pharmaceutically active agents including trastuzumab-MCC-DM1 and aselective Bcl-2 inhibitor selected from the lists of the additionalagents described herein, along with any pharmaceutically inactiveexcipients, diluents, carriers, or glidants. The bulk composition andeach individual dosage unit can contain fixed amounts of the aforesaidpharmaceutically active agents. The bulk composition is material thathas not yet been formed into individual dosage units. An illustrativedosage unit is an oral dosage unit such as tablets, pills, capsules, andthe like. Similarly, the herein-described method of treating a patientby administering a pharmaceutical composition of the present inventionis also intended to encompass the administration of the bulk compositionand individual dosage units.

Pharmaceutical compositions also embrace isotopically-labeled compoundsof the present invention which are identical to those recited herein,but for the fact that one or more atoms are replaced by an atom havingan atomic mass or mass number different from the atomic mass or massnumber usually found in nature. All isotopes of any particular atom orelement as specified are contemplated within the scope of the compoundsof the invention, and their uses. Exemplary isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine,chlorine and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Certainisotopically-labeled compounds of the present invention (e.g., thoselabeled with ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (³H) and carbon-14 (¹⁴C) isotopes areuseful for their ease of preparation and detectability. Further,substitution with heavier isotopes such as deuterium (²H) may affordcertain therapeutic advantages resulting from greater metabolicstability (e.g., increased in vivo half-life or reduced dosagerequirements) and hence may be preferred in some circumstances. Positronemitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸F are useful for positronemission tomography (PET) studies to examine substrate receptoroccupancy. Isotopically labeled compounds of the present invention cangenerally be prepared by following procedures analogous to thosedisclosed in the Schemes and/or in the Examples herein below, bysubstituting an isotopically labeled reagent for a non-isotopicallylabeled reagent.

Trastuzumab-MCC-DM1 and selective Bcl-2 inhibitors may be formulated inaccordance with standard pharmaceutical practice for use in atherapeutic combination for therapeutic treatment (includingprophylactic treatment) of hyperproliferative disorders in mammalsincluding humans. The invention provides a pharmaceutical compositioncomprising trastuzumab-MCC-DM1 in association with one or morepharmaceutically acceptable carrier, glidant, diluent, or excipient.

Suitable carriers, diluents and excipients are well known to thoseskilled in the art and include materials such as carbohydrates, waxes,water soluble and/or swellable polymers, hydrophilic or hydrophobicmaterials, gelatin, oils, solvents, water and the like. The particularcarrier, diluent or excipient used will depend upon the means andpurpose for which the compound of the present invention is beingapplied. Solvents are generally selected based on solvents recognized bypersons skilled in the art as safe (GRAS) to be administered to amammal. In general, safe solvents are non-toxic aqueous solvents such aswater and other non-toxic solvents that are soluble or miscible inwater. Suitable aqueous solvents include water, ethanol, propyleneglycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixturesthereof. The formulations may also include one or more buffers,stabilizing agents, surfactants, wetting agents, lubricating agents,emulsifiers, suspending agents, preservatives, antioxidants, opaquingagents, glidants, processing aids, colorants, sweeteners, perfumingagents, flavoring agents and other known additives to provide an elegantpresentation of the drug (i.e., a compound of the present invention orpharmaceutical composition thereof) or aid in the manufacturing of thepharmaceutical product (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form.

Suitable containers are well known to those skilled in the art andinclude materials such as bottles (plastic and glass), sachets,ampoules, plastic bags, metal cylinders, and the like. The container mayalso include a tamper-proof assemblage to prevent indiscreet access tothe contents of the package. In addition, the container has depositedthereon a label that describes the contents of the container. The labelmay also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration withpharmaceutically acceptable diluents, carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences (1995) 18th edition,Mack Publ. Co., Easton, Pa.), in the form of a lyophilized formulation,milled powder, or an aqueous solution. Formulation may be conducted bymixing at ambient temperature at the appropriate pH, and at the desireddegree of purity, with physiologically acceptable carriers, i.e.,carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8.

The pharmaceutical formulation is preferably sterile. In particular,formulations to be used for in vivo administration must be sterile. Suchsterilization is readily accomplished by filtration through sterilefiltration membranes.

The pharmaceutical formulation ordinarily can be stored as a solidcomposition, a lyophilized formulation or as an aqueous solution.

The pharmaceutical formulations of the invention will be dosed andadministered in a fashion, i.e., amounts, concentrations, schedules,course, vehicles and route of administration, consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat the coagulation factor mediated disorder. Suchamount is preferably below the amount that is toxic to the host orrenders the host significantly more susceptible to bleeding.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl, ethanol, orbenzylalcohol; alkyl parabens such as methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, including Tween 80, PLURONICS™ orpolyethylene glycol (PEG), including PEG400. The active pharmaceuticalingredients may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences18th edition, (1995) Mack Publ. Co., Easton, Pa.

The pharmaceutical formulations include those suitable for theadministration routes detailed herein. The formulations may convenientlybe presented in unit dosage form and may be prepared by any of themethods well known in the art of pharmacy. Techniques and formulationsgenerally are found in Remington's Pharmaceutical Sciences 18^(th) Ed.(1995) Mack Publishing Co., Easton, Pa. Such methods include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

As a general proposition, the initial pharmaceutically effective amountof trastuzumab-MCC-DM1 administered per dose will be in the range ofabout 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

In a preferred embodiment, trastuzumab-MCC-DM1 is formulated as alyophilized powder in single-use vials containing 100 mg per vial or 160mg per vial, and is administered at a dose of 3.6 mg/kg as anintravenous infusion every 3 weeks.

Pharmaceutical compositions of the anti-Bcl-2 active agent alone, e.g.the Bcl-2 inhibitor, depend on their pharmaceutical properties; e.g. forsmall chemical compounds such as e.g. ABT-737, ABT-199 or ABT-263, oneformulation could be e.g. the following:

a) Tablet Formulation (Wet Granulation):

Item Ingredients mg/tablet 1. Compound of formula (I) 5 25 100 500 2.Lactose Anhydrous DTG 125 105 30 150 3. Sta-Rx 1500 6 6 6 30 4.Microcrystalline Cellulose 30 30 30 150 5. Magnesium Stearate 1 1 1 1Total 167 167 167 831

Manufacturing Procedure:

1. Mix items 1, 2, 3 and 4 and granulate with purified water.2. Dry the granules at 50° C.3. Pass the granules through suitable milling equipment.4. Add item 5 and mix for three minutes; compress on a suitable press.

b) Capsule Formulation:

Item Ingredients mg/capsule 1. Compound of formula (I) 5 25 100 500 2.Hydrous Lactose 159 123 148 — 3. Corn Starch 25 35 40 70 4. Talc 10 1510 25 5. Magnesium Stearate 1 2 2 5 Total 200 200 300 600

Manufacturing Procedure:

1. Mix items 1, 2 and 3 in a suitable mixer for 30 minutes.2. Add items 4 and 5 and mix for 3 minutes.3. Fill into a suitable capsule.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interracialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

EXAMPLES Example 1

Expression of Bcl-2 Family Pro-Survival Molecules in T-DM1-ResistantBreast Cancer Cells

Preparation of T-DM1 Resistant Cell Lines

Initially, KPL-4 and BT-474M1 cells were made resistant to T-DM1 bycontinuous culture in the presence of T-DM1, starting at a very lowconcentration of 10 ng/mL, which was gradually increased to 2 μg/mL. Thecells derived are the “resistant pools” which were maintained in culturein 2 μg/mL T-DM1. To derive stably resistant clones, each pool (KPL-4and BT-474M1) was subjected to single cell sorting and cloning. Cloneswere maintained without T-DM1 such that clones that had stableresistance in the absence of T-DM1 could be identified.

TaqMan Analysis

Total RNA was prepared using Qiagen RNeasy Mini kit. Genomic DNA wasremoved by DNase I. Gene expression was quantified using real timequantitative PCR (qPCR or TaqMan). TaqMan One-Step Universal Master Mix(Applied Biosystems) was used for all reactions. The reaction wasperformed in a standard 96-well plate format with ABI 7500 Real-TimeqPCR System. 100 ng total RNA was used as template in each reaction. Fordata analysis, raw Ct was normalized to house-keeping gene HP1BP3.

The results of TaqMan analysis are presented in FIG. 1A. The figureshows that Bcl-2 mRNA expression was increased in T-DM1 resistant KPL-4and T-DM1 resistant BT-474M1 cell lines (normalized to the housekeepinggene HP1BP3) relative to the parent, non-resistant KPL-4 and BT-474M1cells.

Western blot analysis was performed as follows: Cells were lysed incorrected FLAG elution buffer (CFEB) (19.17 mM Tris (pH 7.5), 916.7 μMMgCl2, 92.5 mM NaCl and 0.1% Triton X-100) with protease and phosphataseinhibitors (Roche); in some cases 6 M urea was added. Cleared lysateswere quantitated and equal amounts of proteins were reduced, alkylated,separated by SDS-PAGE, and transferred onto PVDF membranes (Invitrogen)following standard procedures. Western blotting was performed asrecommended by the respective antibody manufacturers. Western blotanalysis shown in FIG. 1B confirms that Bcl-2 is overexpressed in T-DM1resistant KPL-4 and BT-474 cell lines.

Example 2

Cell Proliferation Assay—Parental and T-DM1-Resistant KPL-4 BreastCancer Cell Lines

The cell proliferation assay was performed for 3 days using Cell-TiterGlo reagent. KPL-4 parental breast cancer cells and KPL-4T-DM1-resistant breast cancer cells, prepared as described in Example 1,were treated with T-DM1, GDC-0199 or a combination of T-DM1 and GDC-0199at fixed ratios. Synergy was analyzed using the Chou and Talalay DrugCombination Dose-Effect Analysis with CalcuSyn software in order toobtain a Combination Index “CI” (Chou and Talalay (1984) Adv. EnzymeRegul. 22:27-55). CI values of less than 1 indicate synergy, while CI=1indicated additivity.

Assay conditions: Cells were maintained in Ham's F-12: high glucose DMEM(1:1) supplemented with 10% heat-inactivated fetal bovine serum and 2 mML-glutamine. Cells were plated in 96-well plates (4000 cells per wellfor KPL-4 parental cells; 8000 cells per well for KPL-4 T-DM1 resistantcells) and allowed to adhere overnight at 37° C. in a humidifiedatmosphere of 5% CO₂. Medium was then removed and replaced by freshculture medium containing either T-DM1, GDC-0199, the combination ofboth. Cell Titer-Glo (Promega Corp.) was added to the wells at 3 daysafter drug administration and the luminescent signal was measured usingEnVision Multilabel Plate Reader (PerkinElmer). Combination Index (C.I.)values were generated using CalcuSyn software (Biosoft, Inc)

As shown in FIG. 2, the combination of T-DM1 and GDC-0199 has asynergistic anti-proliferative effect in KPL-4 T-DM1-resistant breastcancer cells.

Example 3

Caspase 3/7 Activation Luminescence Assay—Parental and T-DM1-ResistantKPL-4 Breast Cancer Cell Lines

As noted before, the Bcl-2 family of proteins regulates programmed celldeath triggered by developmental cues and in response to multiple stresssignals. When activated, they can permeabilize the outer membrane ofmitochondria and release pro-apoptogenic factors (e.g. cytochrome C)needed to activate the caspases that dismantle the cell (Wang, K., Genesand Development 15 (2001) 2922-2933; (Adams, 2003 supra); Green, D. R.,and Kroemer, G., Science 305 (2004) 626-629). Thus, activation ofcaspases, such as caspases 3 and 7, indicates induction of apoptosis.

In the present experiment, caspase 3/7 activation luminescence assay wasperformed using the Caspase-Glo® reagent (Promega) essentially followingmanufacturer's instructions. Assays were performed in the same manner asthe viability assays except that drug incubation times were 24 hours andCaspase-Glo 3/7 (Promega) was used to measure apoptosis.

As shown in FIG. 3, right panel, T-DM1-resistant KPL-4 (HER2+) breastcancer cells were re-sensitized to T-DM1 when treated for 24 hours witha combination of T-DM1+GDC-0199, as shown by increased apoptosis(increased caspase 3/7 activation). The same combination showed noeffect in parental cells. It is noted that the results were assessed 24hours after treatment, which is too early for apoptosis induced by T-DM1alone in the parental cell line (left panel).

FIGS. 4A and 4B presents the results of a caspase 3/7 activationluminescence in vitro apoptosis assay measuring activation of caspases 3and 7 in KPL-4 T-DM1-resistant human breast cancer cells relative toparental cells using different concentrations of T-DM1 and GDC-0199,respectively. Increased apoptosis is observed in KPL-4 T-DM1 resistantcells upon the addition of increasing concentrations of GDC-0199 (1, 2.5or 5 μM) to either 0.1 or 1 μg/mL T-DM1. In contrast, T-DM1 inducesrobust apoptosis in KPL-4 parental cells which is not further enhancedby the addition of GDC-199.

FIG. 5A presents the results of a caspase 3/7 activation luminescence invitro apoptosis assay measuring activation of caspases 3 and 7 in Clone#17 T-DM1-resistant KPL-4 human breast cancer cell line treated withT-DM1, GDC-0199 or T-DM1+GDC-0199. Similar to observations with theKPL-4 T-DM1 resistant pool of cells, induction of apoptosis in Clone #17was increased upon the addition of increasing concentrations ofGDC-0199.

FIGS. 6A and 6B show the results of a caspase 3/7 activationluminescence in vitro apoptosis assay measuring activation of caspases 3and 7 in Clone #8 T-DM1 resistant KPL-4 human breast cell line treatedwith 0.1 μg/mL and 1 μg/mL T-DM1 concentrations, respectively, alone orin combination with the indicated concentrations of GDC-0199.

As shown in FIGS. 4A, 4B, 5A, 6A and 6B, the results obtained withdifferent clones of T-DM1-resistant KPL-4 breast cancer cell linesconfirm the enhanced proapoptotic activity of the T-DM-1+GDC-0199combination at various concentrations.

Example 4

Xenograft Studies—KPL-4 T-DM1-Resistant Breast Cancer Cell Lines

For all xenograft studies, three million T-DM1-resistant KPL-4 breastcancer cells were implanted in #2/3 mammary fat pads of femaleSCID-beige mice. When tumors reached a volume of approximately 200 mm³,mice were randomized into treatment groups (n=10 mice pre group): 5mg/kg T-DM1 q3w, 100 mg/kg GDC-0199 qd, combination of the two orvehicle. The results of these xenograft studies for xenografts ofvarious clones of the T-DM1-resistant KPL-4 breast cancer cells areshown in FIGS. 5B and 6C. The results indicate enhanced anti-tumoreffect when T-DM1 and GDC-0199 were used in combination, relative tosingle agent activity.

Example 5

IHC Studies—T-DM1-Resistant KPL-4 Xenograft Tumors

FFPE (formalin-fixed paraffin-embedded) xenograft tumors were sectionedfor analysis of Bcl-2 and HER2 (ErbB2) expression byimmunohistochemistry (IHC), using DAB detection method. Bcl-2 antibodySP66 was obtained from Ventana. Human tonsil sections served as Bcl-2positive controls. Anti-HER2 antibody 4D5 was obtained from Ventana.Human breast cancer cell lines served as positive controls (SK-BR-3 as3+; MDA-MB-361 as 2+; MBA-MB-231 as negative).

FIG. 7A shows the expression of Bcl-2 in formalin-fixedparaffin-embedded T-DM1-resistant KPL-4 xenograft tumor samples (Clones#8 and #17) determined by immunohistochemistry (IHC), using DABdetection method, as described above.

FIG. 7B shows the expression of HER2 (ErbB2) in formalin-fixedparaffin-embedded T-DM1-resistant KPL-4 xenograft tumor samples (Clones#8 and #17) determined by immunohistochemistry (IHC), using DABdetection method, as described above.

Anti-Bcl-2 antibody results: Vehicle groups of each clone showed similarlow frequency of Bcl-2 reactive cells, most often located at theperimeter of tumor lobules (not shown). The frequency and intensity ofthe Bcl-2 signal at the tumor lobule margins in the T-DM1 treated groupswas increased or not changed.

Anti-HER2 antibody results: Vehicle and T-DM I treated tumors in allclones showed very high frequency of HER2 3+IHC. In some T-DM1 treatedtumors, there were regions of weaker HER2 staining (clone #17), mostoften adjacent to the stromal bands surrounding the tumor lobules (notshown).

The Bcl-2 IHC results demonstrate that Bcl-2 expression is maintained inthe T-DM1 resistant clones #8 and #17 when grown as xenograft tumors(FIG. 7A; see also Example 6 which shows very little Bcl-2expression inKPL-4 parental cells by Western blot). Bcl-2 expression in T-DM1-treatedclone #17 is higher than the corresponding vehicle control. FIG. 7Bdepicts HER2 expression as assessed by IHC. In contrast to therelatively lower HER2 expression observed in cells grown in vitro,clones #8 and #17, in both vehicle and T-DM1-treated tumors, show highHER2 expression at the 2+ and 3+level. All clone #8 tumors weredetermined to be 85-95% HER2+ or 3+, with a very low frequency of 2+ or1+tumor cells. Clone #17 tumors were more variable, with 35-75% HER23+cells and 20-65% cells HER2 2+ in the vehicle group.

Example 6

Xenograft Studies—KPL-4 T-DM1-Resistant Breast Tumors

FIG. 8 shows Western blot expression data of Bcl-2, HER2, Bcl-xL and Pgpin KPL-4 T-DM1-resistant clone #17 xenograft tumors treated withGDC-0199, T-DM1 or T-DM-1+GDC-0199. (The three digit numbers above lanes4-19 indicated individual xenograft tumors.) The expression data showthat HER2 and Bcl-2 expression are maintained in all groups, as comparedto the corresponding cells grown in vitro in cell culture.

Example 7

Caspase 3/7 Luminescence and Fluorescence ActivationAssays—T-DM1-Sensitive Breast Cancer Cell Lines

Caspase 3/7 activation luminescence assay was performed as described inExample 3.

The caspase 3 activation fluorescence in vitro apoptosis assay wasperformed using IncuCyte™ reagents and equipment to measure caspaseactivation over time (kinetic analysis) essentially followingmanufacturing instructions.

FIG. 9A presents results of a caspase 3/7 luminescence in vitroapoptosis assay, testing the effect of five separate concentrations ofGDC-0199 (μM) in combination with 9 different concentrations of T-DM1 oncaspase activity in HER2+MDA-MB-361 breast cancer cells, which aresensitive to T-DM1 (naïve). The results demonstrate caspases 3 and 7activation with T-DM1 which is enhanced in a dose-dependent manner withincreasing concentrations of GDC-0199.

FIG. 9B presents the results of a caspase 3 fluorescence in vitroapoptosis assay, testing the effect of three different concentrations ofGDC-0199 (0.63 μM, 1.25 μM, 2.5 μM), alone and in combination with T-DM1(0.1 μg/mL), on caspase activity in HER2+T-DM1 sensitive (naïve)MDA-MB-361 breast cancer cells. The results demonstrate that GDC-0199enhances caspase activation above that induced by T-DM1 alone in a dose-and time-dependent manner, and therefore results in enhanced apoptosiswith all combinations.

FIG. 10A presents the results of a caspase 3/7 luminescence in vitroapoptosis assay, testing the effect of five separate concentrations ofGDC-0199 (μM) in combination with 9 different concentrations of T-DM1 oncaspase activity in T-DM1 naïve HER2+HCC1569 breast cancer cells. Theresults demonstrate that T-DM1 alone does not induce apoptosis butaddition of GDC-0199 results in enhanced caspase activity and henceenhanced apoptosis in all combinations.

FIG. 10B presents the results of a caspase 3 fluorescence in vitroapoptosis assay, testing the effect of three different concentrations ofGDC-0199 (0.63 μM, 1.25 μM, 2.5 μM), alone and in combination with T-DM1(0.1 μg/mL), on caspase activity in HER2+HCC1569 breast cancer cells.The results demonstrate that GDC-0199 enhances caspase activation abovethat induced by T-DM1 alone in a dose- and time-dependent manner, andtherefore results in enhanced apoptosis with all combinations.

These results show that the T-DM1/GDC-0199 combination is also effectivein T-DM1 naïve (i.e. not T-DM1 resistant) cell lines.

Example 8

Xenograft Studies—TDM-1-Sensitive (Naïve) MDA-MB-361 Breast Tumors

Ten million MDA-MB-361 breast cancer cells were implanted into the rightmammary fat pad of female NOD/SCID mice one day after implantation of60-day release 17p-estradiol pellets. When tumors reached a volume ofapproximately 200-300 mm³, mice were randomized into treatment groups(n=10 mice per group) and administered T-DM1 (1, 3, or 7 mg/kg i.v.once), GDC-0199 (100 mg/kg qd×21) or a combination of T-DM1 and GDC-0199as shown in FIG. 11. The results indicate enhanced anti-tumor activityof GDC-0199 with 7 mg/kg T-DM1, relative to single agent activity.

Example 9

Western Analysis: Effects of T-DM1+/−GDC-0199 on Bcl-2 Family MemberProteins in HER2+Breast Cancer Cell Lines

The effect of treatment with T-DM1 (1.25 μg/mL) alone or in combinationwith GDC-0199 (1.25 μM) was studied on various T-DM1 naïve HER2+breastcancer cell lines. The results are shown in FIG. 12. Four out of theeight HER2+breast cancer cell lines tested (BT-474, HCC1569, MDA-361 andZR-75-30) expressed Bcl-2; all eight breast cancer cell lines expressedthe other Bcl-2 family members assessed-Bcl-xL and Mcl-. Three of thecell lines (BT-474, MDA_361 and ZR-75-30) showed phosphorylation ofBcl-2 after T-DM1 treatment, a known effect of exposure to anti-mitoticagents such as T-DM1. As shown in FIGS. 9A, 9B, 10A and 10B, MDA-MB-361and HCC1569 showed enhanced apoptosis when treated with a combination ofT-DM1 and GDC-0199.

T-DM1 (KADCYLA®) exhibits significant clinical benefits in the treatmentof cancer for patients, such as breast cancer patients, who haveprogressed on prior HER2-targeted therapies, such as on treatment withtrastuzumab (HERCEPTIN®). The U.S. Food and Drug Administration approvedKADCYLA® (ado-trastuzumab emtansine), for the treatment of patients withHER2-positive, metastatic breast cancer who previously receivedtreatment with trastuzumab and a taxane. The data presented heredemonstrate that combination treatment with a Bcl (e.g. Bcl-2) inhibitorand T-DM1 significantly improves the efficacy of T-DM1 administered as asingle agent. The results also demonstrate that such combinationtreatment with T-DM I and a Bcl-2 inhibitor is effective both in thetreatment of T-DM1 sensitive (naïve) HER2 positive cancers (e.g. breastcancers) and HER2 positive cancers (e.g. breast cancers) resistant totreatment with T-DM1.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and examples forpurposes of clarity of understanding, it will be readily apparent tothose of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretowithout departing from the spirit or scope of the appended claims.

1-50. (canceled)
 51. A method for the diagnosis of a subject with aHER2-positive tumor as being resistant or susceptible to treatment withan anti-HER2 antibody-drug conjugate, comprising (i) obtaining a tumorsample from said subject, (ii) measuring the expression level of theBcl-2 gene or its product in said tumor sample relative to a controlsample, and (iii) diagnosing said tumor as being resistant to treatmentwith an anti-HER2 antibody-drug conjugate when the measured expressionlevel of Bcl-2 in said tumor sample is at least 2 fold greater than theexpression level in said control sample, or diagnosing said tumor asbeing susceptible to treatment with an anti-HER2 antibody-drug conjugatewhen the measured expression level of Bcl-2 in said tumor sample is lessthan 2 fold greater than the expression level in said control sample.52. The method of claim 51, wherein said subject is a human patient. 53.The method of claim 51, wherein said control sample is a tumor sample ofthe same cell type that is not resistant to treatment with saidanti-HER2 antibody-drug conjugate.
 54. The method of claim 51, whereinthe tumor is breast cancer or gastric cancer.
 55. The method of claim51, wherein the tumor sample is a formalin-fixed, paraffin-embeddedtumor sample.
 56. The method of claim 51, further comprising the step ofmeasuring the expression level of the HER2 gene or its product in saidtumor sample.
 57. The method of claim 51, further comprising the step oftreating said subject with an anti-HER2 antibody-drug conjugate and aselective Bcl-2 inhibitor when the measured expression level of Bcl-2 insaid tumor sample is at least 2 fold greater than the expression levelin said control sample.
 58. The method of claim 51, further comprisingthe step of treating said patient with an anti-HER2 antibody-drugconjugate when the measured expression level of Bcl-2 in said tumorsample is less than 2 fold greater than the expression level in saidcontrol sample.
 59. The method of claim 51, wherein the anti-HER2antibody-drug conjugate is trastuzumab-MCC-DM1.
 60. The method of claim51, wherein the selective Bcl-2 inhibitor is2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)-4-(4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazin-1-yl-N-(3-nitro-4-((tetrahydro-2H-pyran-4-yl)methylamino)phenylsulfonyl)benzamideor a pharmaceutically acceptable salt thereof.